Process for preparing an A2A-adenosine receptor agonist and its polymorphs

ABSTRACT

Disclosed is a synthesis suitable for large scale manufacture of an A 2A -adenosine receptor agonist, and also relates to polymorphs of that compound, and to methods of isolating a specific polymorph.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/333,872, filed Dec. 21, 2011, now U.S. Pat. No. 8,524,883, issued onSep. 3, 2013, which is a continuation of U.S. patent application Ser.No. 12/765,623, filed Apr. 22, 2010, now U.S. Pat. No. 8,106,183, issuedon Jan. 31, 2012, which is a continuation of U.S. patent applicationSer. No. 11/701,699, filed Feb. 2, 2007, now U.S. Pat. No. 7,732,595,issued on Jun. 8, 2010, which claims priority to U.S. Provisional PatentApplication No. 60/801,857, filed May 18, 2006, and to U.S. ProvisionalPatent Application No. 60/765,114, filed Feb. 3, 2006, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a process for the large scalepreparation of an A_(2A)-adenosine receptor agonist, and also relates topolymorphs of that compound, and to methods of isolating a specificpolymorph.

BACKGROUND

Adenosine is a naturally occurring nucleoside, which exerts itsbiological effects by interacting with a family of adenosine receptorsknown as A₁, A_(2A), A_(2B), and A₃, all of which modulate importantphysiological processes. One of the biological effects of adenosine isto act as a coronary vasodilator; this result being produced byinteraction with the A_(2A) adenosine receptor. This effect of adenosinehas been found to be useful as an aid to imaging of the heart, wherecoronary arteries are dilated prior to administration of an imagingagent (for example thallium 201), and thus, by observation of the imagesthus produced, the presence or absence of coronary artery disease can bedetermined. The advantage of such a technique is that it avoids the moretraditional method of inducing coronary vasodilation by exercise on atreadmill, which is clearly undesirable for a patient that has acoronary disease.

However, administration of adenosine has several disadvantages.Adenosine has a very short half life in humans (less than 10 seconds),and also has all of the effects associated with A₁, A_(2A), A_(2B), andA₃ receptor agonism. Thus the use of a selective A_(2A) adenosinereceptor agonist would provide a superior method of producing coronaryvasodilation, particularly one with a longer half life and few or noside effects.

A class of compounds possessing these desirable properties was disclosedin U.S. Pat. No. 6,403,567, the complete disclosure of which is herebyincorporated by reference. In particular, one compound disclosed in thispatent,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,has been shown to be a highly selective A_(2A)-adenosine receptoragonist, and is presently undergoing clinical trials as a coronaryvasodilator useful in cardiac imaging.

Given the heightened interest in this and similar compounds, it hasbecome desirable to find new methods of synthesis that provide aconvenient method for making large quantities of the material in goodyield and high purity. The patent that discloses the compound ofinterest (U.S. Pat. No. 6,403,567) provides several methods forpreparing the compound. However, although these methods are suited tosmall scale syntheses, all synthetic methods disclosed in the patentutilize protecting groups, which is undesirable for large scalesyntheses.

Additionally, it was discovered that the desired product (that is(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide)is capable of existing in at least three different crystalline forms,the most stable of which is a monohydrate. This polymorph is stableunder relative humidity stress conditions, up to its melting point.Accordingly, it is desirable that the final product produced in the newsyntheses is obtained as the stable monohydrate.

SUMMARY OF THE INVENTION

Thus, it is an object of this invention to provide convenient synthesesfor the large scale preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,and polymorphs thereof, preferably as its monohydrate. Accordingly, in afirst aspect, the invention relates to the preparation of a compound ofthe Formula I:

comprising:contacting a compound of the formula (3):

with methylamine.

In one embodiment the reaction is conducted in an aqueous solution ofmethylamine, initially at a temperature of about 0-5° C., followed bywarming to about 50-70° C. Alternatively, the reaction is conducted asabove but in a sealed pressure reactor.

In a second embodiment, the product is isolated as the pure monohydrateby dissolving the product in a solvent, for example dimethylsulfoxide,addition of purified water, filtering the slurry thus formed, washingthe contents of the filter with water followed by ethanol, and dryingthe solid that remains under vacuum at a temperature that does notexceed 40° C.

In a second aspect, the invention relates to the preparation of acompound of the formula (3):

comprising:contacting a compound of the formula (2):

with ethyl 2-formyl-3-oxopropionate.

In one embodiment, the reaction is conducted in ethanol, at atemperature of about 80° C., with about 1.1 molar equivalents of ethyl2-formyl-3-oxopropionate.

In a third aspect, the invention relates to the preparation of acompound of the formula (2):

comprising:contacting a compound of the formula (1):

with hydrazine.

The above described synthesis is suitable for the large scale synthesisof the desired product, which is provided in good yield, although oneminor impurity is seen in the final product. This impurity has beenshown to be unchanged intermediate of the formula (2); that is, thecompound of the formula:

Although this impurity can be removed from the final product bycrystallization, it was decided to seek an alternative synthesis thathad all of the advantages of the above synthesis but did not give thecompound of formula (2) as an impurity in the final product.

Thus, in a fourth aspect, the invention relates to a method ofsynthesizing(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideby contacting a compound of the formula (4):

with methylamine.

In one embodiment the reaction is conducted in an aqueous solution ofmethylamine, initially at a temperature of about 0-5° C., followed bywarming to about 50-70° C. Preferably, the reaction is conducted in asealed pressure reactor.

In a second embodiment, the product is isolated as the pure monohydrateby dissolving the product in a solvent, for example dimethylsulfoxide,addition of purified water, filtering the slurry thus formed, washingthe contents of the filter with water followed by ethanol, and dryingthe solid that remains under vacuum at a temperature that does notexceed 40° C.

In a fifth aspect, the invention relates to a method of synthesizing acompound of the formula (4):

comprising contacting a compound of the formula (2):

with an excess of ethyl 2-formyl-3-oxopropionate, preferably about a2-10 fold excess, more preferably about a 5-10 fold excess.

In one embodiment, the reaction is conducted in ethanol, at atemperature of about 80° C. The ethyl 2-formyl-3-oxopropionate ispresent in a 5-10 fold excess.

DEFINITIONS AND GENERAL PARAMETERS

FIG. 1 is a ¹H NMR spectrum of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate (Form A).

FIG. 2 shows the thermal analysis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate.

FIG. 3 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate.

FIG. 4 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideForm B.

FIG. 5 shows the X-Ray diffraction pattern for(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideForm C as compared to Form A.

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

The term “therapeutically effective amount” refers to that amount of acompound of Formula I that is sufficient to effect treatment, as definedbelow, when administered to a mammal in need of such treatment. Thetherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, which can readily be determined by one of ordinary skill inthe art.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   -   (i) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   (ii) inhibiting the disease, that is, arresting the development        of clinical symptoms; and/or    -   (iii) relieving the disease, that is, causing the regression of        clinical symptoms.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The term “polymorph” is intended to include amorphous and solvates of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide.

It has been discovered that this compound is capable of existing in atleast three different crystalline forms, referred to herein as Form A,Form B, Form C, and an amorphous product.

Form A:

This polymorph can be produced by crystallizing1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidefrom protic solvents, for example ethanol or ethanol/water mixtures, orfrom a polar solvent, for example dimethylsulfoxide/water. Form A hasbeen shown to be a monohydrate, and is the most stable of the variouspolymorphs at ambient temperatures. It is stable under relative humiditystress conditions up to its melting point.

Form B:

This polymorph is produced by evaporating under vacuum a solution of1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidein trifluoroethanol at ambient temperatures. The X-ray analysis of thecrystals was distinctly different from any other polymorph (see FIG. 4),but it was difficult to determine its constitution, as the X-rayanalysis gave disordered broad peaks, and the polymorph containedvarying amounts of water. It was found to be difficult to reliablyreproduce the preparation of this polymorph.

Form C:

This polymorph is produced by slurrying1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidein acetonitrile for a long period of time at 60° C. The X-ray analysisof the crystals was distinctly different from any other polymorph (seeFIG. 5). Polymorph C was shown to be a variable hydrate, which, atelevated temperatures, desolvates to an unstable form.

Amorphous Material:

This polymorph is produced by heating Form A polymorph at a temperatureof up to 200° C. This polymorph is unstable in the presence ofatmospheric moisture, forming variable hydrates.

Techniques for Analysis of Forms A, B, C and Amorpheous Material

X-Ray Powder Diffraction

X-ray powder diffraction (XRPD) analyses were carried out on a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation. Theinstrument was equipped with a fine focus X-ray tube, and the tubevoltage and amperage were set to 40 kV and 40 mA respectively. Thedivergence and scattering slits were set at 1″ and the receiving slitwas set at 0.15 mm. Diffracted radiation was detected by a NaIscintillation detector. A theta-two theta continuous scan at 3°/min (0.4sec/0.02° step) from 2.5-40° 2θ was used. A silicon standard was used tocheck the instrument alignment. Data were collected and analyzed usingXRD-6000 v.4.1 software.

X-ray powder diffraction (XRPD) analyses were also performed using anInel XRG-3000 diffractometer equipped with a CPS (Curved PositionSensitive) detector with a 28 range of 120°. The instrument calibrationwas performed using a silicon reference standard. The tube voltage andamperage were set to 40 kV and 30 mA, respectively. The monochromatorslit was set at 5 mm by 80 μm. Samples were placed in an aluminum sampleholder with a silicon insert or in glass XRPD-quality capillaries. Eachcapillary was mounted onto a goniometer head that is motorized to permitspinning of the capillary during data acquisition. Real time data werecollected using Cu-Kα radiation at a resolution of 0.03° 20. Typically,data were collected over a period of 300 seconds. Only the data pointswithin the range of 2.5-40° 20 are displayed in the plotted XRPDpatterns.

Thermal Analyses

Thermogravimetric (TG) analyses were carried out on a TA Instruments2050 or 2950 thermogravimetric analyzer. The calibration standards werenickel and Alumel™. Samples were placed in an aluminum sample pan,inserted into the TG furnace, and accurately weighed. The samples wereheated in nitrogen at a rate of 10° C./min to either 300 or 350° C.Unless stated otherwise, samples weights were equilibrated at 25° C. inthe TGA furnace prior to analysis.

Differential scanning calorimetry (DSC) analyses were carried out on aTA Instruments differential scanning calorimeter 2920. Accuratelyweighed samples were placed in either crimped pans or hermeticallysealed pans that contained a pinhole to allow for pressure release. Eachsample was heated under nitrogen at a rate of 10° C./min to either 300or 350° C. Indium metal was used as the calibration standard.Temperatures were reported at the transition maxima.

Infrared Spectroscopy

Infrared spectra were acquired on Magna 860® Fourier transform infrared(FT-IR) spectrophotometer (Nicolet Instrument Corp.) equipped with anEver-Glo mid/far IR source, an extended range potassium bromidebeamsplitter, and a deuterated triglycine sulfate (DTGS) detector.Unless stated otherwise, a Spectra-Tech, Inc. diffuse reflectanceaccessory (the Collector™) was used for sampling. Each spectrumrepresents 256 co-added scans at a spectral resolution of 4 cm⁻¹. Samplepreparation for the compound consisted of placing the sample into amicrocup and leveling the material with a frosted glass slide. Abackground data set was acquired with an alignment mirror in place. Thespectra represent a ratio of the sample single-beam data set to thebackground single beam data set. Wavelength calibration of theinstrument was performed using polystyrene.

NMR Spectroscopy

Solution phase ¹H NMR spectra of the were acquired at ambienttemperature on a Bruker model AM-250 spectrometer operating at 5.87 T(Larmor frequency: ‘H=250 MHz). Time-domain data were acquired using apulse width of 7.5 ps and an acquisition time of 1.6834 second over aspectral window of 5000 Hz. A total of 16,384 data points werecollected. A relaxation delay time of 5 seconds was employed betweentransients. Each data set typically consisted of 128 coaveragedtransients. The spectra were processed utilizing GRAMS132 A1 software,version 6.00. The free induction decay (FID) was zero-filled to fourtimes the number of data points and exponentially multiplied with aline-broadening factor of 0.61 Hz prior to Fourier transformation. The‘H spectra were internally referenced to tetramethylsilane (0 ppm) thatwas added as an internal standard.

Alternatively, NMR analysis was carried out as described in Example 4.

Moisture Sorption/Desorption Analyses

Moisture sorption/desorption data were collected on a VTI SGA-100 VaporSorption Analyzer. Sorption and desorption data were collected over arange of 5% to 95% relative humidity (RK) at 10% RH intervals under anitrogen purge. Sodium chloride (NaCl) and polyvinyllpyrrolidone (PVP)were used as the calibration standards. Equilibrium criteria used foranalysis were less than 0.0100% weight change in 5 minutes, with amaximum equilibration time of 180 minutes if the weight criterion wasnot met. The plotted data have not been corrected for the initialmoisture content.

NOMENCLATURE

The structure of the compound(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis as follows:

Synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide

One method for the large scale synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis shown in Reaction Scheme I.

Step 1—Preparation of Formula (2)

The compound of formula (2) is prepared from the compound of formula (1)by reaction with hydrazine monohydrate in the absence of a solvent. Thereaction is conducted at a temperature of about 40° C. plus/minus 5° C.When the reaction is complete, the product of formula (2) is isolated bystirring with a protic solvent in which the compound of formula (2) haslimited solubility, for example ethanol or isopropanol. The mixture isstirred for about 1-5 hours, and then filtered. The solid is purified bystirring with water, filtering, and washing with water followed byisopropanol and dried under vacuum, which is taken to the next stepwithout purification.

Step 2—Preparation of Formula (3)

The compound of formula (2) is then converted to a compound of formula(3) by reacting with about 1-1.2 molar equivalents of ethyl2-formyl-3-oxopropionate. The reaction is conducted in a protic solvent,preferably ethanol, at about reflux temperature, for about 2-4 hours.After cooling, to about 0° C., the solid is filtered off, washed withcold ethanol, and dried under reduced pressure. The product of formula(3) is taken to the next step without purification.

Step 3—Preparation of Final Product

The final product is prepared from the compound of formula (3) byreacting with methylamine, preferably aqueous methylamine. The reactionis carried out at about room temperature, for about 4 hours. The productof Formula I is isolated by conventional means, for example byfiltration, washing the solid with cold ethanol, and drying underreduced pressure.

Preparation of Starting Materials

(4S,2R,3R,5R)-2-(6-amino-2-chloropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diolis used as a starting material in step 1. This compound is commerciallyavailable.

Ethyl 2-formyl-3-oxopropanoate is used as a starting material in step 2.It is commercially available, or may be made as shown in Reaction SchemeII.

Ethyl 3,3-diethoxypropionate is reacted with ethyl formate in thepresence of a strong base, preferably sodium hydride. The reaction iscarried out at about 0-5° C., for about 24 hours. The product isisolated by conventional means, for example by the addition of water andextraction of impurities with a conventional solvent, for examplet-butylmethyl ether, acidification of the aqueous phase with, forexample, hydrochloric acid, followed by extraction with a solvent suchas dichloromethane, and removing the solvent from the dried extractunder reduced pressure.

A preferred method for the large scale synthesis of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideis shown in Reaction Scheme III.

Step 1—Preparation of Formula (2)

The compound of formula (2) is prepared from the compound of formula (1)by reaction with hydrazine monohydrate in the absence of a solvent. Thereaction is conducted at a temperature of about 45-55° C. plus/minus 5°C. When the reaction is complete, the product of formula (2) is isolatedby stirring with a protic solvent in which the compound of formula (2)has limited solubility, for example ethanol or isopropanol. The mixtureis stirred for about 1-5 hours, and then filtered. The solid is purifiedby stirring with water, filtering, and washing with water followed byethanol or isopropanol and dried under vacuum, which is taken to thenext step without purification.

Step 2—Preparation of Formula (4)

The compound of formula (2) is then converted to a compound of formula(4) by reacting with an excess of ethyl 2-formyl-3-oxopropionate, forexample a 2-10 fold excess, preferably about 5-10 fold excess. Thereaction is conducted in a protic solvent, for example ethanol, at aboutreflux temperature, for about 2-4 hours. After cooling, to about 0° C.,the solid is filtered off, washed with cold ethanol, and dried underreduced pressure, and the product of formula (4) is taken to the nextstep without purification.

The compound of formula (4) is drawn as a (2E) alkene derivative, asthis is the major isomer formed in this reaction. However, it should benoted that a significant amount of the (2Z) alkene derivative may alsobe formed in this reaction; that is:

named as ethyl(2Z)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate.

Accordingly, although the compound of formula (4) is represented as the(2E) alkene derivative only, the term “compound of formula (4)” isintended to include both the instance where it is solely the (2E)isomer, and the instance where the major portion of the product is the(2E) isomer and a minor portion of the (2Z) isomer is also present. Theconversion of the compound of formula (4) to the final product byreaction with methylamine as described in Step 3 proceeds in the samemanner whether the compound of formula (4) is present as the (2E) isomeror as a mixture of the (2E) isomer and the (2Z) isomer.

Step 3—Preparation of Final Product

The final product is prepared from the compound of formula (4) byreacting with methylamine, preferably aqueous methylamine. The reactionis initially carried out at about 0-5° C. for about 8 hours, preferablyin a pressure reactor, followed by raising the temperature to 50-60° C.over about 1 hour, and maintaining the temperature for 15-30 minutes.The product is isolated by conventional means, for example by cooling to0-5° C. and maintaining a vacuum for about 1 hour, thus removing themethylamine. The vacuum is removed, and the remaining contents held at0-5° C. for at least 30 minutes, followed by filtration. The solid thusobtained is washed with water followed by ethanol, and dried underreduced pressure.

This process provides(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate. This polymorph can be further purified by dissolvingin dimethylsulfoxide, filtering any solid impurities from the solution,and precipitating the monohydrate from solution by addition of water.

EXAMPLE 1 Preparation of Ethyl-2-formyl-3-oxopropionate

A three- or four-neck round bottom flask equipped with magnetic stirbar, thermocouple, digital thermometer, gas inlet and outlet andaddition funnel was flushed with argon. Ethyl 3,3-diethoxypropionate(64.5 g) in tetrahydrofuran were charged to the addition funnel. Sodiumhydride (21.2 g of a 60% dispersion) was charged to the reaction flaskfollowed by tetrahydrofuran. The contents of the flask were cooled to0-5° C. in an ice-bath, and ethyl formate (257 g) was added. The mixturewas cooled to 0-5° C. and the contents of the addition funnel addeddropwise, maintaining an internal temperature of less than 5° C. Theice-bath was removed and the contents allowed to warm to ambienttemperature. Consumption of ethyl 3,3-diethoxypropionate was monitoredby TLC analysis. The reaction was quenched by addition of ice-water(10.6 vol), and extracted three times with methyl t-butyl ether (5.4 voleach), and the organic layers discarded. The aqueous phase was acidifiedwith conc. hydrochloric acid to a pH of 1 to 1.5. The acidified aqueouslayer was extracted three times with dichloromethane and the combinedorganic layers dried over sodium sulfate. The solvent was removed underreduced pressure, and the residue distilled under vacuum, to provideethyl 2-formyl-3-oxopropionate, 27.92 g, 70% yield.

EXAMPLE 2 A. Preparation of 2-Hydrazinoadenosine (2)

A flask equipped with a mechanical stirrer, gas inlet, gas outlet andthermocouple was flushed with argon. 2-Chloroadenosine hemihydrate (53.1g) was added, followed by hydrazine monohydrate (134 g). The mixture wasstirred while heating to 40-45° C. for 2 hours. The progress of thereaction was followed by TLC analysis. When the reaction was complete,the heat source was removed and ethanol (800 ml) was added. The mixturewas stirred for 2 hours at ambient temperature, then the precipitatecollected by filtration. The filter cake was washed with ethanol anddried under reduced pressure for 30 minutes. The solids were transferredto a clean flask equipped with a mechanical stirrer and water (300 ml)was added. The suspension was stirred at room temperature for 18 hours,and the solids isolated by filtration. The filter cake was washed withice-cold water (300 ml) followed by a wash with ice-cold ethanol (300ml). The solid was dried under reduced pressure to provide2-hydrazinoadenosine (41.38 g, 81.4% yield, 99.3% purity).

B. Alternative Preparation of 2-Hydrazinoadenosine (2)

A reaction vessel containing hydrazine hydrate (258 g, 250 ml) washeated to 40-50° C. To the warm mixture 2-chloroadenosine hemihydrate(100 g) was added in portions, maintaining the temperature between45-55° C. The temperature was kept at this temperature for two hours,and then deionized water (500 ml) was added over a period of 30 minutes,maintaining the temperature at 45-55° C. The mixture was then graduallycooled to 0-5° C. over a period of 3 hours, then stirred at thistemperature for a further 30 minutes. The solid was then filtered off,and washed with cold (2-5° C.) deionized water (200 ml), followed byethanol (400 ml). The solid was dried under vacuum for 12 hours, toprovide 2-hydrazinoadenosine.

EXAMPLE 3 Preparation of Ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate(3)

Ethyl 2-formyl-3-oxopropionate (23.93 g, 0.17 mol) was placed in a flaskequipped with mechanical stirrer, gas inlet, gas outlet and refluxcondenser. 2-Propanol was added to the flask followed by2-hydrazinoadenosine (44.45 g, 0.15 mol). The mixture was heated toreflux under stirring for 2-4 hours, following the progress of thereaction by TLC analysis. When the reaction was judged complete, theheat source was removed and the mixture cooled to room temperature. Thesuspension was cooled under stirring in an ice-bath for 1.5 to 2 hours.The solids were isolated by vacuum filtration, and washed with ice-cold2-propanol. The product, ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate,was dried under reduced pressure to a constant weight. Yield 54.29 g,purity (by HPLC) 96.6%.

EXAMPLE 4 Preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide

A mixture of ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate(46.4 g) and methylamine (40% in water, 600 ml) was stirred at ambienttemperature for about 4 hours, following the progress of the reaction byHPLC analysis. The majority of the excess methylamine was removed underreduced pressure, and the remaining mixture cooled at 0° C. for 2 hours.The solid material was filtered off, washed with ice-cold 200 proofethanol, and dried under reduced pressure, to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate, 36.6 g, purity 99.6%.

The structure of the material was confirmed by ¹H NMR (see FIG. 1 andbelow). Thermal analysis (see FIG. 2) provided results consistent withthe presence of one molecule of water. X-Ray powder diffraction patternswere obtained (FIG. 3)

¹H and ¹³C NMR spectra were obtained in the following manner. Twosamples of the material obtained above were weighed out and dissolved ind₆-DMSO—5.3 mg was used for the ¹H spectra, and 20.8 mg was used for ¹³Cspectra. All spectra were acquired at ambient temperature on a JEOLEclipse⁺ 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for¹³C.

Multiplicity, Label ¹³C shift (ppm) ¹H shift (ppm) splitting (Hz) 2150.5 or 150.3 — 4 156.4 — 4a 117.9 — 6 140.0 8.41 s 7a 150.5 or 150.3 —1′ 86.9 5.94 D, 6.2 2′ 73.7 4.62 m 2′-OH — 5.50 D, 6.2 3′ 70.5 4.17 m3′-OH — 5.23 D, 4.7 4′ 85.7 3.96 m 5′ 61.5 3.67, 3.57 m 5′-OH — 5.02 D,5.7 A 140.9 8.07 D, 0.8 B 120.2 — C 129.6 8.95 D, 0.8 D 161.7 — E 25.62.76 D, 4.6 NH₂ — 7.77 br s NH — 8.35 Q, 4.6

Purification of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate

A solution of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate (100 g) in dimethylsulfoxide (300 ml) was filtered through a0.6 to 0.8 micron prefilter and a 0.2 micron filter to remove any solidimpurities. The filtrate was then slowly added over a period of 1 hourto deionized water (1 liter) with stirring, and the slurry thus producedstirred for not less than 1 hour. The solid was filtered off, washedwith deionized water (2×1 liter), and dried under vacuum for not lessthan 1 hour. The dried product was then slurried again with deionizedwater (1.5 liter) for not less than 2 hours, filtered off, and washedwith deionized water (1 liter) followed by absolute ethanol (750 ml).The purified product was dried under vacuum at a temperature of not morethan 40° C. for not less than 12 hours, to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidemonohydrate free of any 2-hydrazinoadenosine impurity.

EXAMPLE 5 Preparation of Ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate

A mixture of 2-hydrazinoadenosine (100 g, 0.34 mol), ethyl2-formyl-3-oxopropionate (242 g, 1.7 mol) and absolute ethanol werecharged to a reactor, and the mixture heated to reflux for 2 hours. Whenthe reaction was judged complete, the heat source was removed and themixture gradually cooled to 5-10° C. over a period of 3 hours. Theslurry was stirred for 30 minutes at this temperature, and the mixturefiltered. The solid material was washed with cold (5-10° C.) absoluteethanol, and then dried under vacuum at a temperature that did notexceed 40° C., to provide ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-[4-(ethoxycarbonyl)-pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate.

An elemental analysis gave the following results: C, 48.75%; H, 4.86%;N, 18.05%; O, 27.57. Theoretical: C, 49.72%; H, 4.74%; N, 18.45%; O,27.09. The analysis corresponds within experimental error limits to thehemihydrate of the desired product. (C, 48.89%; H, 4.81%; N, 18.1%; O,28.12)

¹H and ¹³C NMR spectra were obtained in the following manner. 20.2 mg ofthe compound of formula (4) was dissolved in −0.75 ml of DMSO-d6, andthe spectra obtained at ambient temperature on a JEOL ECX-400 NMRspectrometer operating at 400 MHz for ¹H and 100 MHz for ¹³C. Thechemical shifts were referenced to the DMSO solvent, 2.50 ppm for ¹H and39.5 ppm for ¹³C.

Results

The ¹H and ¹³C chemical shifts are listed in Table 1. Two isomers in aratio of ˜60/30 were observed in both the ¹H and the ¹³C spectra,labeled as major and minor in the table.

¹³C Chemical ¹H Chemical Multiplicity^(b), Atom^(a) Shift (ppm) Shift(ppm) Splitting (Hz) 21(major) 192.4 9.96 d, 3.6 21(minor) 187.6 9.83 S22(minor) 167.1 — — 22(major) 165.2 — — 15(minor) 161.8 — 15(major)161.7 — —  6(major) 153.1 — —  6(minor) 152.9 — —  2(minor) 149.4 — — 2(major) 149.3 — — 19(minor) 148.0 9.22 d, 13.0  4(minor) 147.9 — — 4(major) 147.8 — — 19(major) 147.5 9.26 d, 12.4, d, 3.6  8(major) 144.98.87 s  8(minor) 144.7 8.85 s 12 143.1 8.20-8.23 m 14(minor) 132.8 9.20d, ~0.7 14(major) 132.6 9.12 d, ~0.7  5(major) 120.7 — —  5(minor) 120.6— — 13 116.7 — — 20(minor) 107.2 — — 20(major) 106.1 — —  1′ (major)87.9 6.07 d, 5.3  1′ (minor) 87.9 6.06 d, 5.3  4′ 85.8 4.02 q, 3.9 2′(minor) 74.1 4.62 q, ~5.4  2′(major) 74.1 4.61 q, ~5.4  3′ 70.1 4.22q, 4.2  5′ 61.0 3.62, 3.73 m 23, 16 60.3-60.8 4.25-4.39 m 17, 2414.1-14.2 1.28-1.38 m 18(major) — 12.51  d, 12.4 18(minor) — 11.47  d,13.0  2′-OH (major) — 5.63 d, 6.1  2′-OH (minor) — 5.62 d, 6.1  3′-OH —5.30 d, 5.1  5′-OH — 5.08 t, 5.5The compound of formula (4) was confirmed to be a mixture of thefollowing two isomers:

EXAMPLE 6 Preparation of(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamidefrom Compound (4)

Aqueous 40% methylamine solution (1300 ml) was placed in a pressurereactor, cooled to 0-5° C., and the product of Example 5 (ethyl(2E)-3-({9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-[4-(ethoxycarbonyl)pyrazolyl]purin-6-yl}amino)-2-formylprop-2-enoate(100 g) added. The mixture was stirred at 0-5° C. for at least 8 hours,monitoring the reaction for completion. When complete, the mixture waswarmed, maintaining the temperature between 50 and 60° C. for 1 hour,and then cooled to less than 30° C. over a period of 1 hour. When thetemperature was below 30° C., the mixture was degassed using a pressureof 100-150 mm Hg, allowing the temperature to decrease to 0-5° C. Themixture was stirred at 0-5° C. for at least 1 hour, maintaining thepressure at 100-150 mm Hg. The vacuum was then discontinued and replacedby nitrogen, maintaining the temperature at 0-5° C. for not less than 30minutes. The solid product was then filtered off, washed with water(3×500 ml), then with absolute ethanol (625 ml). The product was driedunder vacuum, not allowing the temperature to exceed 40° C., to provide(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideas its monohydrate.

¹H and ¹³C NMR spectra were obtained in the following manner. Twosamples of the material obtained above were weighed out and dissolved ind₆-DMSO—5.3 mg was used for the ¹H spectra, and 20.8 mg was used for ¹³Cspectra. All spectra were acquired at ambient temperature on a JEOLEclipse⁺ 400 spectrometer operating at 400 MHz for ¹H and 100 MHz for¹³C.

Multiplicity, Label ¹³C shift (ppm) ¹H shift (ppm) splitting (Hz) 2150.5 or 150.3 — 4 156.4 — 4a 117.9 — 6 140.0 8.41 s 7a 150.5 or 150.3 —1′ 86.9 5.94 D, 6.2 2′ 73.7 4.62 m 2′-OH — 5.50 D, 6.2 3′ 70.5 4.17 m3′-OH — 5.23 D, 4.7 4′ 85.7 3.96 m 5′ 61.5 3.67, 3.57 m 5′-OH — 5.02 D,5.7 A 140.9 8.07 D, 0.8 B 120.2 — C 129.6 8.95 D, 0.8 D 161.7 — E 25.62.76 D, 4.6 NH₂ — 7.77 br s NH — 8.35 Q, 4.6

An elemental analysis gave the following results: C, 43.96%; H, 4.94%;N, 27.94. Theoretical: C, 44.12%; H, 4.94%; N, 27.44%; O, 27.09. Theanalysis corresponds within experimental error limits to themonohydrate.

We claim:
 1. A pharmaceutical composition prepared from a crystallinemonohydrate form of the compound(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamideby adding at least one pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein the monohydrate issubstantially free of 2-hydrazinoadenosine.
 3. The pharmaceuticalcomposition of claim 2, wherein the monohydrate is substantially free ofother hydrates or amorphous form.
 4. The pharmaceutical composition ofclaim 3, wherein the monohydrate has a purity of at least about 99.6%.5. The pharmaceutical composition of claim 4, wherein the monohydrateexhibits an X-ray powder diffraction pattern having peaks at diffractionangle 2θ (°) of about 6, about 9, about 11, about 13, about 14.5, about16.5, and about 18 as measured by Cu-Kα1 X-ray powder diffractometry.